The generation of 1000s of patient exomes and genomes, while accelerating diagnoses, has also highlighted the complexity of previously-considered ?simple? traits. Numerous examples are now reported of patients with deleterious alleles in multiple genes. In some, the phenotype reflects an amalgam of two disorders; in others, it is the product of genetic interactions. Moreover, recent exome- or panel-based resequencing of groups of genes known to cause a host of dominant or recessive disorders are beginning to report an enrichment of rare variants in patients, highlighting the concept of mutational burden. Bardet-Biedl syndrome (BBS), a founding ciliopathy, has been a model for studying these phenomena and for beginning to understand the contribution of such alleles to non-penetrance and variable expressivity. This is because: (a) the majority of the recessive burden in BBS is now known, with ~80% of BBS patients harboring recessive mutations in 22 genes; (b) most BBS proteins are necessary for cilia structure/function and assemble into defined complexes; and (c) we and others have developed quantitative in vitro and in vivo tools to assess the total functional output of the cilium; to establish the effect of variants; and to measure genetic interactions. This Renewal focuses on three themes. First, we will capitalize on extensive genetic and functional data from previous cycles to build a comprehensive map of mutational distribution in a biological module. Through the rigorous analysis of two independent BBS patient cohorts, we have observed a 2.5-fold increase of rare variants in known BBS genes beyond the recessive driver. This variation is not distributed randomly but intimates an interaction between mutations that map to different macromolecular complexes. Using established, mouse models of BBS, we will ask how these interactions might potentiate or exacerbate discrete BBS endophenotypes. Second, we will take advantage of recent observations in humans and mouse models of ciliopathies to dissect the role of cis- and -trans acting genetic modifiers. Focusing on TTC21B/IFT139, a gene that contributes causal and modifying mutations across the ciliopathy disease spectrum, we will test the hypothesis that mutations in that locus contribute to the development of renal disease in BBS, by leveraging extensive data from zebrafish models and testing the paradigm in the mouse. In parallel, using a combination of computational and biological tools, will ask whether discrete point mutations in that locus can account for the variable activity of the disease-causing allele p.P209L, a hypomorph in human but a null allele in the mouse. Finally, grounded on recent observations derived from a genome-wide genetic suppressor screen that augmentation of the proteasome ameliorates BBS in vitro and in vivo, we will employ our recently-developed proteasome sensor screening paradigm to perform a small molecule screen for new therapeutic leads. These studies will provide a comprehensive look at the architecture of a genetically heterogeneous, phenotypically variable exemplar disorder, while at the same time, allowing the rational discovery of new therapeutic paradigms.